Isoprenoids are isoprene polymers that find use in pharmaceuticals, neutraceuticals, flavors, fragrances, and rubber products. Natural isoprenoid supplies, however, are limited due to ecological concerns. For this reason, and to provide isoprenoid compositions having fewer impurities and greater uniformity, isoprenoids such as rubber are often produced synthetically.
Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in water and soluble in alcohol. Commercially viable quantities of isoprene can be obtained by direct isolation from petroleum C5 cracking fractions or by dehydration of C5 isoalkanes or isoalkenes (Weissermel and Arpe, Industrial Organic Chemistry, 4th ed., Wiley-VCH, pp. 117-122, 2003). The C5 skeleton can also be synthesized from smaller subunits. It would be desirable, however, to have a commercially viable method of producing isoprene that was independent of nonrenewable resources.
Biosynthetic production of isoprene occurs by two distinct metabolic pathways (Julsing et al., Appl Microbiol Biotechnol, 75:1377-1384, 2007). In eukaryotes and archae, isoprene is formed via the mevalonate (MVA) pathway, while some eubacteria and higher plants produce isoprene via the methylerythritol phosphate (MEP) pathway. Isoprene emissions from plants are light and temperature-dependent with increases linked to leaf development. An isoprene-producing enzyme, isoprene synthase, has been identified in Aspen trees (Silver and Fall, Plant Physiol, 97:1588-1591, 1991; and Silver and Fall, J Biol Chem, 270:13010-13016, 1995) and is believed to be responsible for the in vivo production of isoprene from whole leaves. Bacterial production of isoprene has also been described (Kuzma et al., Curr Microbiol, 30:97-103, 1995; and Wilkins, Chemosphere, 32:1427-1434, 1996), and varies in amount with the phase of bacterial growth and the nutrient content of the culture medium (U.S. Pat. No. 5,849,970 to Fall et al.; and Wagner et al., J Bacteriol, 181:4700-4703, 1999, both herein incorporated by reference in their entirety). The levels of isoprene obtainable through bacterial systems of the prior art, however, are insufficient for commercial uses.
Polypeptides, e.g. isoprene synthase, have a three-dimensional structure determined by the primary amino acid sequence and the environment surrounding the polypeptide. This three-dimensional structure establishes the polypeptide's activity, stability, binding affinity, binding specificity, and other biochemical attributes. Thus, knowledge of a protein's three-dimensional structure can provide much guidance in designing improvements to its biological activity, for example, greater catalytic activity, dimerization and/or solubility.
The three-dimensional structure of a polypeptide can be determined in a number of ways. Many of the most precise methods employ X-ray crystallography (See, e.g., Van Holde, (1971) Physical Biochemistry, Prentice-Hall, New Jersey, pp. 221-39). This technique relies on the ability of crystalline lattices to diffract X-rays or other forms of radiation. Diffraction experiments suitable for determining the three-dimensional structure of macromolecules typically require high-quality crystals. The crystallization properties of a polypeptide vary greatly (Dale, et al., J. Struct. Biol. 142:88-97, 2003; MacPherson, A., Methods 34:254-265, 2004; and Slabinski, L et al., Protein Science 16:2472-2482, 2007). In some cases polypeptides crystallize readily whereas in other cases polypeptides have proven extremely difficult to obtain. There is no comprehensive theory to guide efforts to crystallize macromolecules and as a result, most efforts macromolecular crystal growth is empirical in nature (MacPherson, 2004).
Previous efforts to utilize the structure of isoprene synthase in order to improve production of isoprene have relied on the structures of other terpene synthases in which three-dimensional structures are available including bornyl diphosphate synthase and 5-epi-aristolochene synthase (See e.g., U.S. patent application Ser. No. 12/429,143 and WO 2008/137092). What is needed is a three-dimensional structure of isoprene synthase to aid in the design of variants of isoprene synthase to allow commercial scale biological production of isoprenoids.
A three-dimensional structure of isoprene synthase is found to have a structurally homologous fold with previously determined synthetases, e.g. bornyl diphosphate synthase and limonene synthase, including conservation in the region involved with the coordinates of required metal ion cofactors, the active and substrate binding sites. The structure has provided insight in the conformational changes that are necessary for these enzyme to bind substrate and catalyze a coordinated series of reactions. Specifically the structure has identified flexible regions that are likely to be shared by all structurally homologous synthetase and that modification of the amino acids found in these regions and the neighboring regions would be expected to effect improved performance of these enzymes. A three-dimensional structure of isoprene synthase may provide a three-dimensional configuration of points, or reaction coordinates, representing the active site of isoprene synthase for the conversion of dimethylallyl diphosphate (DMAPP) into isoprene. Such a configuration of points can aid in the design of synthetic agents for the conversion of DMAPP into isoprene.